Helicobacter pylori (Hp) is a major cause of peptic ulcer disease and an early risk factor for gastric cancer. Metronidazole (Mtz) is an antibacterial agent used against Hp, but resistance is common and is a major reason for treatment failure. Research by D. Berg has shown that this resistance is a polygenic trait and that null mutations in the gene rdxA confers low-level resistance; a mutation in another gene, frxA, results in higher resistance and that higher resistance (hyper-resistance) results from mutations in another genes that remain to be identified (Jeong et al. J. Bact. 182:3219, 2000). We [Pan and Weissman PNAS: 99:9346 (2002)] have developed a method for global screening for mutations in complex DNA mixtures. In the present application we propose to optimize and apply this method for analysis of bacterial genomes, and use the method to identify mutations and genes that make Hp hyper-resistant to Mtz. The final purpose of this project is to provide a general research tool for dissecting the molecular mechanisms of bacterial pathogenicity and genome evolution. These experiments will help meet Specific Aim 4 of parent grant AI38166 to DE Berg (co-PI) [To more fully understand mechanisms of drug resistance]. This project is "exploratory" or "developmental," relative to the parent grant, in that we may need to overcome potential "noise" from duplicate and divergent sequences as well as other unexpected problems, and generally optimize this for microbial systems. Nevertheless, this method should allow identification of the genes responsible for hyper-resistance with an efficiency that could not be matched by traditional (e.g. shotgun cloning and DNA transformation) type protocols, especially if different constellations of genes cause hyperR in different strains (genetic backgrounds).